Skin is a complex, multi-layered, large, and visible organ of the mammalian (e.g., human) body that is important to health and, for self-aware mammals such as humans, self-image. Skin comprises three principal layers (i.e., the epidermis, the dermis, and the hypodermis or subcutis) that contain a variety of cell types and structures, including epidermal and dermal connective tissue with blood and lymphatic vessels, hypodermal adipose tissue, and elastic fascia beneath the hypodermis. In turn, these structures are composed of a number of different cell types including keratinocytes, melanocytes, fibroblasts, endothelial cells, and adipocytes.
The skin functions, in part, as an immune organ, acting as a barrier against environmental factors and mounting an inflammatory response to such factors, when necessary, in order to protect against injury and infection. In this role, skin brings to bear several types of immune cells such as macrophages, dendritic cells, mast cells, and T-cells. These cells provide not only protective immunosurveillance against foreign objects such as microbes, they also release cellular factors to maintain homeostasis, including factors that regulate inflammation of the skin. Various factors involved in regulating skin inflammation (e.g., interleukins), promote homeostasis in response to a variety of health-related conditions, such as eczema, psoriasis, wounds, bacterial exposure, and dandruff. Moreover, the inflammatory response mounted by skin can arise in different ways. For example, the release of pro-inflammatory cytokines can induce inflammation in skin, as can a mechanical breach of skin integrity. Although skin inflammation can arise in a number of ways, however, these inflammatory responses do share common characteristics at both the molecular and physiological level.
Given the undesirable impact that skin-related inflammation can have on one's health, appearance and self-esteem, there is continuing interest in identifying cosmetic and/or therapeutic agents that are effective for treating or improving the appearance of skin by modulating skin inflammation. Skin models capable of more closely mimicking the in vivo skin condition would be expected to increase the accuracy of identifying modulators of skin inflammation.
Modeling techniques to study skin physiology and skin responses to agents have historically included a variety of specific techniques, from the culturing of a single cell type or a small number of commingled cell types, to fabricating tissue equivalents, to developing animal models. These relatively simple models, however, often lack entire skin layers, or particular skin cell types or structures, or intra- and/or inter-cellular communications found with skin in the in vivo condition. For example, cell cultures of single cell types are readily available but lack connections to other features of skin and often exhibit behavior in culture that is not seen in the in vivo skin condition. Additionally, such cells may be altered by genetic manipulation to promote cell passaging and maintenance.
More complex skin models include animal models and skin-equivalent models. While having additional complexity, animal models can suffer from limitations including genetic differences relative to human skin. Results obtained with animal models, moreover, are burdened with the concern that human skin tissues might react differently.
Skin-equivalent models can be limited by lack of cellular interconnectivity, permeability concerns, and anatomical simplicity. Some recent attempts at skin-equivalent models may be described as organotypic human tissue equivalents and include in vitro reconstructions of human cells such as keratinocytes cultured on an inert polycarbonate filter. These models, by their nature, are limited in that they can have reduced barrier function that can lead to aberrant sensitivities to tested agents. The models may also be less complex than human skin, having perhaps one or two cells types (such as keratinocytes and fibroblasts or keratinocytes and melanocytes), but lacking additional cells such as endothelial cells or even the full keratinocyte, fibroblast, and melanocyte combination. In addition, the organotypic skin-equivalent models may also be missing normal skin structures, such as glands, that may affect skin response.
The most complex skin model involves the ex vivo culture of human skin tissue samples. Some previous attempts at such models included small biopsies of skin floating directly in culturing media. It is known in the art that transient cultures may be deficient, as inventors and researchers have indicated attempts at ex vivo pig skin grafts are limited to seven days (Vielhaber et al., Ex vivo Human Skin Model, US2009/0298113). Attempts at improving the longevity of ex vivo skin have been sought, one example being described in EP 2 019 316 B1. Three-dimensional culture of skin cells has also been attempted, but those efforts focused on culturing immature fetal cells that had markedly different compositions from mature human skin in terms of the relative frequency of stem cells, the level of damaged cells, and the extent of senescent cells within the culture. Collectively, these differences establish the 3-D cultures as distinct from cultures of mature in vivo skin.
Even more recent ex vivo skin models have involved attempting to culture skin explants on metal grids (Mitts et al., Elastin Protective Polyphenolics and Methods of Using the Same, US2009/0110709) and skin grafting to the chorioallantoic membrane (CAM) of a fertilized ovarian egg (Goldstein et al., Chimeric Avian-Based Screening System Containing Mammalian Grafts, US2009/0064349). Such models, however, remain vulnerable to transient lifespans, excessive sensitivity to environmental influences (e.g., nutrient availability, toxin concentrations, heat), and the aforementioned limits on the ability to extrapolate results across species boundaries.
Beyond the difficulties in developing a skin model that accurately and precisely mimics the in vivo skin condition in efforts to identify agents such as modulators of skin inflammation, a method for identifying such modulators must identify controls that behave predictably with the skin model and in vivo skin. In addition, for screens relying on the larger inflammatory responses associated with induced inflammation, it is difficult to identify a level of inducer that will result in a significant inflammatory response without unacceptable additional effects such as the induction of proliferation. For example, IL-22 is an inflammation inducer that is also an inducer of cell proliferation. Depending on dosage and time, such inducers provoke a thickened hyperplastic response in skin samples, progressively destroying the integrity of the skin sample. Further, for screens designed to allow an inflammatory state to stabilize or normalize, inducer administrations (quantity and frequency) need to be compatible with extended culturing periods.
In view of the foregoing observations, it is apparent that human skin inflammation is associated with a variety of diseases and disorders, and there remains a need in the art for improved methods of identifying modulators of skin inflammation as well as the modulators themselves and methods of using such modulators to treat, prevent, or ameliorate a symptom associated with skin inflammation, or simply to improve the cosmetic appearance of skin. A need also exists for identifying conditions under which organ samples suitable for use in modeling inflammation, such as ex vivo skin samples can be maintained and/or grown in culture for extended times without significant deviation from the behavior of such materials in vivo.